Structure/function analysis of the Drosophila fat facets deubiquitinating enzyme and analysis of the fat-dependent signaling pathway

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Abstract

I am interested in a cell communication pathway that determines cell fate in
Drosophila eye. This pathway, in particular, is mediated by a deubiquitinating
enzyme (DUB) called Fat facets (Faf). Further studies revealed another key
component, Liquid facets (Lqf), which is a Drosophila homolog of epsin. Epsin in
vertebrates is known to function in clathrin-mediated endocytosis. Thus, Ub
pathway and endocytosis are both essential for cell patterning in Drosophila eye
development. My goal is to understand the role of Faf in cell fate determination in
the eye. First, I performed a structure/function analysis of Faf protein in three
different ways: (1) Deleted forms of the faf gene were generated in vitro and
introduced into the fly genome to express partial Faf proteins. Each of the 6 deleted
Faf proteins was tested for its ability to complement the faf mutant eye phenotype
in vivo; (2) The DNA sequence aberrations in 14 mutant faf alleles were determined
and these data defined specific domains and amino acids required for the normal
function of Faf; (3) The homolog of faf in mouse, Fam, was tested for its activities
in Drosophila. I found that Fam is able to substitute faf in all of its essential
functions in vivo, indicating that they are indeed functional homologs. In addition,
all Drosophila UBPs were retrieved from Genome Project and aligned for any
conserved functional motifs (this part of work is done by J. Fischer). DUBs are a
large family of proteins about which little is known. This part of my work
contributes to a better understanding of DUB function.
The second part of my thesis is to analyze the faf-dependent signaling
pathway. (1) The lqf mutation was identified in an enhancer screen for faf
mutations. To identify more components of this pathway, a former graduate
student, Qinghong Li, performed a screen for suppressors of the faf mutant eye
phenotype. Two suppressors were found to have essential functions by themselves.
One mutation is in UbcD1 which encodes an E2. I analyzed the second suppressor
and discovered that it is allelic to DNAprim, which encodes the 60-kD subunit of
DNA polymerase. I think that the interaction between DNAprim and faf is likely to
be indirect. Overall, this part of my work contributes to better understanding the
faf-dependent pathway in eye development; (2) Genetic experiments performed by
a former graduate student, Angelica Cadavid, indicated Lqf is a candidate substrate
of Faf in the eye. But there is no biochemical evidence so the conclusion is highly
speculative. In collaboration with Dr. Bing Zhang, we generated antibodies to Lqf,
and I used it in a variety of biochemistry experiments. The data are consistent with
our genetic results. For the first time, a DUB is demonstrated to regulate the
ubiquitination state of a specific substrate and this event is critical to a cell
communication pathway in development.